NEUTRON STUDIES OF REGULATION OF VERTEBRATE MUSCLE

脊椎动物肌肉调节的中子研究

• 批准号: 2909829
• 负责人: ROBERT A MENDELSON
• 金额: $ 29.17万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2909829
• 关键词: actins; calcium ion; conformation; laboratory rabbit; microfilaments; muscle contraction; neutron diffraction; nuclear magnetic resonance spectroscopy; protein structure function; structural biology; tropomyosin; troponin

Vertebrate muscle contraction is regulated by the binding and unbinding of Ca2+ to the thin-filament protein troponin (TN). Upon Ca2+ binding, structural changes occur within troponin, which are transmitted to the thin- filament protein tropomyosin (Tm), which runs along the helical groove of actin. Tm, in turn, is believed to mediate the interaction of myosin and actin, which is responsible for muscle contraction. We propose to use neutron scattering, as well as an NMR experiment, to elucidate the in situ structures and organization of Tn subunits within whole Tn with and without regulating Ca2+ bound to TnC. The spatial organization of Tn subunits will be studied with respect to Tm and the complete thin filament. Deuteration of a member of a complex will allow member(s) to be rendered "invisible" to neutrons. Structures of the visible components will be thus investigated in situ without ambiguity. Specifically, we will study the in situ structures of the Tn subunits TnC, TnI and TnT+/- regulatory Ca2+. We will determine the organization of Tn subunits +/-Ca2+ in whole troponin as well as in thin filaments. We will determine the radial position of Tn subunits relative to Tm +/- Ca2+. A search for changes in the radial and azimuthal positions of Tn subunits +/-Ca2+ in solutions and oriented thin filaments will help us to further understand how troponin subunits control Tm movement during regulation. We will also investigate the changes in the in situ structures of F- actin as a "control" for these experiments. We propose implementing a novel NMR method for determining the high-resolution in situ structure of TnC. This proposal is directed at fundamental basic science questions concerning molecular aspects of the regulation of skeletal muscle contraction. However, this structural information may give insight into disease processes such as hypertrophic cardiomyopathies which are associated with missence mutations in cardiac troponins I and T and tropomyosin. It is anticipated that these same techniques may in the future be applied to the study of cardiac regulatory proteins and may provide a structural basis for understanding the role of phosphorylation of cardiac TnI, the unique features of the interaction of cardiac TnI with cardiac TnC (which possesses only a single regulatory Ca2+-binding site), and the effect of ischemia and calcium-sensitizing drugs on TnC.

脊椎动物的肌肉收缩是通过 Ca2+ 与细丝蛋白肌钙蛋白 (TN) 的结合和解除结合来调节的。 Ca2+ 结合后，肌钙蛋白内发生结构变化，并传递至细丝蛋白原肌球蛋白 (Tm)，该蛋白沿着肌动蛋白的螺旋沟延伸。反过来，Tm 被认为介导肌球蛋白和肌动蛋白的相互作用，从而导致肌肉收缩。我们建议使用中子散射以及核磁共振实验来阐明整个 Tn 内 Tn 亚基的原位结构和组织，无论是否调节与 TnC 结合的 Ca2+。 Tn 亚基的空间组织将根据 Tm 和完整的细丝进行研究。复合体成员的氘化将使成员对中子“不可见”。因此，将在原位毫无疑问地研究可见成分的结构。具体来说，我们将研究 Tn 亚基 TnC、TnI 和 TnT+/- 调节 Ca2+ 的原位结构。我们将确定整个肌钙蛋白以及细肌丝中 Tn 亚基 +/-Ca2+ 的组织。我们将确定 Tn 亚基相对于 Tm +/- Ca2+ 的径向位置。寻找溶液和定向细丝中 Tn 亚基 +/-Ca2+ 的径向和方位角位置的变化将有助于我们进一步了解肌钙蛋白亚基在调节过程中如何控制 Tm 运动。我们还将研究 F-肌动蛋白原位结构的变化作为这些实验的“对照”。我们建议实施一种新的 NMR 方法来确定 TnC 的高分辨率原位结构。该提案针对有关骨骼肌收缩调节的分子方面的基本科学问题。然而，这种结构信息可能有助于深入了解与心肌肌钙蛋白 I 和 T 以及原肌球蛋白的错误突变相关的肥厚型心肌病等疾病过程。预计这些相同的技术将来可能会应用于心脏调节蛋白的研究，并可能为了解心脏 TnI 磷酸化的作用、心脏 TnI 与心脏 TnC（仅具有单个调节 Ca2+ 结合位点）相互作用的独特特征以及缺血和钙敏药物对 TnC 的影响提供结构基础。